Biospecific binding reactants labeled with new luminescent lanthanide chelates and their use

ABSTRACT

This invention relates to a luminescent lanthanide chelate comprising a lanthanide ion and a chelating ligand of formula (I)                  
 
wherein
 
     R 1  is selected from the group consisting H, —COOH, —COO − , —CH 2 COOH and —CH 2 COO − ; G 1  is a group consisting of one or two moieties each moiety being selected from the group consisting of ethynediyl, ethenylene, phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, fyrazanylene, 1,2,4-triazol-3,5-ylene and oxadiazolylene; G 2  for coupling to a biospecific binding reactant is selected from the group consisting of amino, aminooxy, carbonyl, aldehyde or mercapto groups and activated forms made of them; Z is selected from the group consisting of carboxyalkyl amine, ether, thioether, carbonyl and unsubstituted or substitute methyl (—CR 2 —) wherein group R 2  is selected from the group consisting of H, methyl, ethyl and carboxylalkyl; and the lanthanide ion is europium(III), terbium(III), dysprosiym(III) or samarium(III). This invention further relates to a detectable molecule comprising the lanthanide chelate and the use of the molecule in a method of carrying out a biospecific binding assay.

FIELD OF THE INVENTION

The present invention relates to detectable molecules comprisinglanthanide chelates attached to a biospecific binding reactant and useof said detectable molecules in bioaffinity based binding assays. Theinvention further relates to new luminescent lanthanide chelates usefulin the preparation of said detectable molecules.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

In specific binding assays, such as, e.g. immunoassays, DNAhybridization assays, receptor-binding assays, and cellular bindingassays, generally the analytes to be measured are present at very lowconcentrations. Therefore, various labeling compounds have beendeveloped that allow the labeling reactant to be detected and quantifiedat high sensitivity. In immunoassays and DNA hybridization assaystime-resolved luminescence spectroscopy using lanthanide chelates iswell known (e.g. I. Hemmilä, T. Stålberg, and P. Mottram (eds.),“Bioanalytical Applications of Labelling Technologies”, Wallac, Turku,1994 and D. Wild (eds), “The Immunoassay Handbook”, Nature PublishingGroup, 2001). Stable photoluminescent (referred in the context of thisspecification simply as luminescent) lanthanide chelates also have otherapplications, e.g. fluorescence microscopy and cytometry. Therefore, anumber of attempts have been made to develop new highly luminescentchelates suitable for those types of time-resolved fluorometricapplications. These include e.g. stable chelates composed of derivativesof pyridine (U.S. Pat. No. 4,920,195; U.S. Pat. No. 4,801,77; U.S. Pat.No. 4,761,481; U.S. Pat. No. 5,571,897; U.S. Pat. No. 5,859,215; Latva,M., Takalo, H., Mukkala, V. M., Matachescu, C., Rodriquez-Ubis, J. C.and Kankare, J., 1997, J. Luminescence, 75, 149; Takalo, H., Hemmilä,I., Sutela, T. and Latva, M., 1996, Helv. Chim. Acta, 79, 789),bipyridines (U.S. Pat. No. 5,216,134), terpyridines (U.S. Pat. No.4,859,777, U.S. Pat. No. 5,202,423, U.S. Pat. No. 5,324,825) or variousphenolic compounds (U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191, IT42508A/89) as energy mediating groups and polycarboxylic acids aschelating parts. In addition, various dicarboxylate derivatives (U.S.Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563),macrocyclic cryptates (U.S. Pat. No. 4,927,923, PCT WO 93/5049, EP 0 493745), calixarenes (Sato, N. and Shinkai, S., 1993, J. Chem. Soc. PerkinTrans. 2, 621; Steemers, F. J., Verboom, W., Reinboudt, D. N., van derTol, E. B. and Verhoeven, J. W., 1995, J. Am. Chem. Soc., 117, 9408),DTPA carbostril 124 conjugate (Selvin, P. R., Rana, T. M. and Hearst, J.E., 1994, J. Am. Chem. Soc., 116, 6029) and macrocyclic Schiff bases (EP0 369 000) have been disclosed in patent applications and/or patents.

It is known that the luminescence lanthanide chelates are quenched in anaqueous solution. When water molecules are coordinated in the innersphere of chelates, quenching is a result of an efficient, radiationlessdecay process involving vibronic coupling of lanthanide excited stateand OH oscillation. The process is additive in regard to the number ofOH oscillators, and hence the luminescence decay is inversely related tothe number of bound water molecules. Various systems have been developedto avoid this phenomenon, such as using detergents and synergisticcompounds, using high concentration of fluorine ions, removing water bydrying prior to measurement, using a polymetric matrix, or measuring theluminescence in an organic solvent or in deuterium oxide. An ideal wayto avoid direct aqueous quenching is to use stable, preferable ninedentate chelating agents, which do not allow the coordination of waterwith the chelated ion. In the above-mentioned chelates the lanthanideion is normally coordinated to 7, 8 or 9 heteroatoms forming a seven-,eight- or nine-dentate chelate, respectively. Seven- and eight-dentatechelates contain from two to one water molecules and thus suffer aqueousquenching. It's generally assumed, that additional coordination atoms innine—dentate chelates—having no water molecule in the first coordinationsphere—don't have any additional positive effect in relation to aqueousquenching.

During an energy transfer process from an excited ligand to a lanthanideion the energy undergoes intersystem crossing to one of ligand tripletstates. The next step is a spin-forbidden transition of the energy,causing ligand phosphorescence, or an intra-molecular energy transfer tothe lanthanide ion. Thermal decay, such as e.g. molecule thermalmovement and rotation, is a known non-radiative deactivation process ofmentioned triplet state. The lanthanide label chelates normally containsone reactive functional group for coupling the label to a biospecificbinding reactant. Thus, in a labeled biomolecule the label may rotateand non-radiative deactivation of ligand triplet state is a possiblephenomenon.

The general view is that several reactive binding groups in a labelmolecule cause cross reaction and formation of the biospecific bindingreactant aggregates during the labeling process, and thus producepurification problems and decreased yield of labeled material.

SUMMARY OF THE INVENTION

An object of this invention is to provide an improved luminescentlanthanide chelate comprising a lanthanide ion and a chelating ligand.

Another object of the present invention is to provide an improveddetectable molecule comprising a biospecific binding reactant attachedto the luminescent lanthanide chelate.

Yet another object of this invention is to provide an improved labelingmethod of a biospecific binging reactant by using the luminescentlanthanide chelate label.

Thus according to one aspect this invention provides a luminescentlanthanide chelate comprising a lanthanide ion and a chelating ligand ofthe formula (I)

wherein,

-   -   a) R₁ is selected from the group consisting of H, —COOH, —COO⁻,        —CH₂COOH and —CH₂COO⁻;    -   b) G₁ is a group consisting of one or two moieties each moiety        being selected from the group consisting of ethynediyl (—C≡C—),        ethenylene (—CH═CH—), phenylene, biphenylene, naphthylene,        pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene,        furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene,        thiazolylene, isothiazolylene, oxazolylene, isoxazolylene,        fyrazanylene, 1,2,4-triazol-3,5-ylene and oxadiazolylene;    -   c) G₂ for coupling to a biospecific binding reactant is selected        from the group containing amino, aminooxy, carbonyl, aldehyde or        mercapto groups or an activated form made of them;    -   d) Z is selected from the group consisting of carboxyalkyl        amine, ether, thioether, carbonyl and unsubstituted or        substitute methyl wherein the group R₂ is selected from a group        consisting of H, methyl, ethyl and carboxylalkyl; and    -   e) the lanthanide ion is europium(III), terbium(III),        dysprosium(III) or samarium(III).

According to another aspect the present invention provides a detectablemolecule comprising a biospecific binding reactant attached to aluminescent lanthanide chelate comprising a lanthanide ion and achelating ligand of the formula (I)

wherein,

-   -   a) R₁ is selected from the group consisting of H, —COOH, —COO⁻,        —CH₂COOH and —CH₂COO⁻;    -   b) G₁ is a group consisting of one or two moieties, each moiety        being selected from the group consisting of ethynediyl,        ethenylene, phenylene, biphenylene, naphthylene, pyridylene,        pyrazinylene, pyrimidinylene, pyridazinylene, furylene,        thienylene, pyrrolylene, imidazolylene, pyrazolylene,        thiazolylene, isothiazolylene, oxazolylene, isoxazolylene,        fyrazanylene, 1,2,4-triazol-3,5-ylene and oxadiazolylene;    -   c) G₂ for coupling to a biospecific binding reactant is selected        from the group consisting of thiourea, aminoacetamide, amide,        aliphatic thioether, disulfide or        6-substituted-1,3,5-tiazine-2,4-diamine;    -   d) Z is selected from the group consisting of carboxyalkyl        amine, ether, thioether, carbonyl and unsubstituted or        substitute methyl wherein group R₂ is selected from the group        consisting of H, methyl, ethyl and carboxylalkyl; and    -   e) the lanthanide ion is europium(III), terbium(III),        dysprosium(III) or samarium(III).

According to yet another aspect the present invention provides animproved labeling method of a biospecific binging reactant. The methoduses the luminescent lanthanide chelate label of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a purification profile of a protein labeled with aeuropium(III) chelate of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention providing a nine- or ten-dentatelanthanide chelate with two binding groups decreases aqueous quenchingeven compared to prior art seven to nine-dentate chelates. Anon-radiative deactivation process of the triplet state caused by labelrotation is prevented, and a more luminescent lanthanide chelate is madepossible, and moreover at the same time, all other important features oflabels and labeled biomolecules can be retained without any additionalforming of aggregates and purification problems.

The aim of the present invention is to provide means to obtain improvedlanthanide chelate labels to be used in specific bioaffinity basedbinding assays, such as immunoassays (both heterogeneous and homogenousassays), DNA hybridization assays, receptor binding assays,immunocytochemical or immunohistochemical assays utilizing fluorometricor time-resolved fluorometric determination of the specificluminescence.

The chelates of this invention combine several important features in asingle label, such as

-   -   1. high absorptivity at suitable wavelength, preferable over 300        nm,    -   2. several separate UV absorbing parts (chromophors) in the same        ligand structure, preferable two chromophors,    -   3. effective energy transfer from the UV absorbing part (triplet        sensitizer) to the lanthanide ion,    -   4. a strongly chelating part to create a) thermodynamic        stability required for storing the labeled reactants for        extended periods of time, and b) high kinetic stability to allow        the use of reactants in conditions where competing metal ions or        chelating agents may be present,    -   5. a chelating part forming as complete a protection of the        chelated ion as possible, preferable a nine-dentate ligand, and        more preferable a ten-dentate ligand,    -   6. a functional group allowing effective coupling of the chelate        to be used as a binding reactant (e.g. antibody) without        destroying its binding properties and decreasing the        luminescence properties of the label, preferable two said        functional groups preventing label rotation after coupling to a        biospecific binging reactant.

In addition, the chelate has to be highly hydrophilic and possess lownonspecific binding affinity to proteins or surfaces used in theanalysis.

The present invention provides a luminescent lanthanide chelatecomprising a lanthanide ion and a chelating ligand of the formula (I)

wherein,

-   -   a) R₁ is selected from the group consisting of H, —COOH, —COO⁻,        —CH₂COOH and —CH₂COO⁻;    -   b) G₁ is a group consisting of one or two moieties each moiety        being selected from the group consisting of ethynediyl (—C≡C—),        ethenylene (—CH═CH—), phenylene, biphenylene, naphthylene,        pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene,        furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene,        thiazolylene, isothiazolylene, oxazolylene, isoxazolylene,        fyrazanylene, 1,2,4-triazol-3,5-ylene and oxadiazolylene;    -   c) G₂ for coupling to a biospecific binding reactant is selected        from the group containing amino, aminooxy, carbonyl, aldehyde or        mercapto groups or an activated form made of them such as        isocyanato, isothiocyanato, diazonium, bromoacetamido,        iodoacetamido, reactive esters, pyridyl-2-dithio or        6-substituted 4-chloro-1,3,5-triazin-2-ylamino;    -   d) Z is selected from the group consisting of carboxyalkyl amine        {—N[(CH₂)_(n)COOH]— or —N[(CH₂)_(n)COO⁻]— and n=1, 2, 3, 4, 5 or        6}, ether (—O—), thioether (—S—), carbonyl (—CO—) and        unsubstituted or substitute methyl (—CR₂—) wherein the group R₂        is selected from a group consisting of H, methyl, ethyl and        carboxylalkyl [—(CH₂)_(n)COOH or —CH₂)_(n)COO⁻ and n=1, 2, 3, 4,        5 or 6]; and    -   e) the lanthanide ion is europium(III), terbium(III),        dysprosium(III) or samarium(III).

The present invention also provides a detectable molecule comprising abiospecific binding reactant attached to a luminescent lanthanidechelate comprising a lanthanide ion and a chelating ligand of formula(I)

wherein,

-   -   a) R₁ is selected from the group consisting of H, —COOH, —COO⁻,        —CH₂COOH and —CH₂COO⁻;    -   b) G₁ is a group consisting of one or two moieties, each moiety        being selected from the group consisting of ethynediyl (—C≡C—),        ethenylene (—CH═CH—), phenylene, biphenylene, naphthylene,        pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene,        furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene,        thiazolylene, isothiazolylene, oxazolylene, isoxazolylene,        fyrazanylene, 1,2,4-triazol-3,5-ylene and oxadiazolylene;    -   c) G₂ for coupling to a biospecific binding reactant is selected        from the group consisting of thiourea (—NH—CS—NH—),        aminoacetamide (—NH—CO—CH₂—NH—), amide (—NH—CO—, —CO—NH—,        —NCH₃—CO— and —CO—NCH₃—), aliphatic thioether (—S—), disulfide        (—S—S—) or 6-substituted-1,3,5-triazine-2,4-diamine;    -   d) Z is selected from the group consisting of carboxyalkyl amine        {—N[(CH₂)_(n)COOH]— or —N[(CH₂)_(n)COO⁻]— and n=1, 2, 3, 4, 5 or        6}, ether (—O—), thioether (—S—), carbonyl (—CO—) and        unsubstituted or substitute methyl (—CR₂—) wherein group R₂ is        selected from the group consisting of H, methyl, ethyl and        carboxylalkyl [—(CH₂)_(n)COOH or —(CH₂)_(n)COO⁻ and n=1, 2, 3,        4, 5 or 6]; and    -   e) the lanthanide ion is europium(III), terbium(III),        dysprosium(III) or samarium(III).

This invention further relates to the use of a detectable molecule asdefined above in biospecific binding assays.

The biospecific binding reactant is selected from a group consisting ofan antibody, an antigen, a receptor ligand, a specific binding proteinor peptide, and a DNA- or RNA-probe.

The substituents in 6-substituted-1,3,5-triazine-2,4-diamine and6-substituted 4-chloro-1,3,5-triazin-2-ylamino can be selected from thegroup consisting of H, halogen, alkoxy, aryloxy, amino, lower alkyl,substituted amino and thioesters, and preferable selected from the groupconsisting of chloro, fluoro, ethoxy, 2-methoxyethoxy, 2-cyanoethoxy,2,2,2-trifluoroethoxy, thiophenoxy and ethoxycarbonylyhiomethoxy.

The term “luminescent” shall in this invention be understood to mean“photoluminescent” as already stated above.

According to a preferable embodiment of the invention the lanthanide ionis either europium(III) or terbium(III).

The invention is further exemplified by the following examplesdemonstrating the effect of prevented label rotation by two bindinggroups together with a ten-dentate chelate structure on luminescenceintensity and decay times.

The structures and the synthetic route employed in the experimental partare shown in reaction scheme 1 a and 1 b. The schemes illustrate thesynthesis of compound 5 exemplified by examples 1 to 5. Scheme 2illustrates the structures of reference label compounds 6 and 7.Compound 6 is a seven-dentate europium(III) label. Compound 7 is anine-dentate europium(III) label with two separate chromophors and onebinding group. The purification profile, shown in FIG. 1, of a labelprotein according to the invention demonstrates low aggregation and theabsence of labeling chelate, and thus shows that the protein labeledwith a compound of the present invention, i.e. by using several reactivebinding groups in a label molecule, does surprisingly not causepurification problems nor decrease yield of labeled material.

EXAMPLES Example 1

The synthesis of tetra(tert-butyl)2,2′,2″,2′″-{[(ethoxycarbonyl)methylimino]bis(methylene)bis(4-bromopyridine-6,2-diyl)bis(methylenenitrilo)}tetrakis(acetate)(1)

Di(tert-butyl)2,2′-{[4-bromo-6-(bromomethyl)pyridin-2-yl]methylenenitrilo}bis-(acetate)(2.04 g, 4.014 mmol) was dissolved in dry acetonitrile (40 ml). To themixture was added ethyl glycinate hydrochloride (0.28 g, 2.006 mmol) anddiisopropylethylamine (2.8 ml, 16.07 mmol). After stirring for 4 hoursat 48° C., the mixture was evaporated. The residue was dissolved inchloroform (120 ml) and washed twice with water (2×30 ml), dried withsodium sulfate and evaporated. The product was purified with flashchromatography (silica, first 20% ethyl acetate in petroleum ether andfinally 50%) The yield was 0.82 g (43%). ¹H NMR(CDCl₃, 400 MHz): 7.75(2H, s), 7.63 (2H, s), 4.1 (2H, q, J=7.04 Hz), 4.00 (4H, s), 3.93 (4H,s), 3.37 (10H, s), 1.46 (36H, s), 1.29 (3H, t, J=7.04 Hz).

Example 2

The synthesis of tetra(tert-butyl)2,2′,2″,2′″-{[(ethoxycarbonyl)methylimino]bis-(methylene)bis{[4-[(4-amino)phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetate)(2)

A mixture of compound 1 (0.3 g, 0.313 mmol),bis(triphenylphosphine)palladium(II)chloride (8.8 mg, 0.0126 mmol) andcopper(I) iodide (4.8 mg, 0.0252 mmol) in dry triethylamine (4 ml) andtetrahydrofuran (4 ml) was deaerated with argon.[(4-Amino)phenyl]acetylene (88 mg, 0.751 mmol) was added to the mixtureand the reaction was stirred overnight at 51° C., after which it wasfiltered and the filtrate evaporated. The residue was dissolved inchloroform (45 ml) and washed twice with water (2×15 ml), dried withsodium sulfate and evaporated. The product was purified with flashchromatography (silica, first from 1% to 10% methanol indichloromethane, and finally methanol) The yield was 0.24 g (75%). ¹HNMR(CDCl₃, 400 MHz): 7.56 (2H, s), 7.53 (2H, s) 7.32 (4H, d, J=8.56 Hz),6.58 (4H, d, J=8.56 Hz), 4.17 (2H, q, J=7.2 Hz)), 4.01 (4H, s), 3.97(4H, s), 3.48(10H, s), 1.46 (36H, s), 1.28 (3H, t, J=7.2 Hz).

Example 3

The synthesis of2,2′,2″,2′″-{[(ethoxycarbonyl)methylimino]bis(methylene)-bis{[4-[(4-amino)phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis-(aceticacid) (3)

A solution of compound 2 (0.1 g, 0.097 mmol) in trifluoroacetic acid (2ml) was stirred for 2 hours at room temperature. After evaporationwithout heating, the mixture was triturated with diethylether (10 ml)and filtered. Filtration left a pure product. The yield was 0.13 g. ¹HNMR(DMSO, 400 MHz): 7.32 (4H, s), 7.11 (4H, d, J=8.3 Hz), 6.42 (4H, d,J=8.3 Hz), 3.94 (2H, q, J=7.1 Hz), 3.82 (8H, s), 3.37 (10H, s), 1.06(3H, t, J=7.1 Hz).

Example 4

The synthesis of{2,2′,2″,2′″-{[(carboxymethyl)imino]bis(methylene)-bis{[4-[(4-amino)phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}-europium(III)(4)

A mixture of compound 3 (180 mg, 0.164 mmol), 0.5 M potassium hydroxidein ethanol (10 ml) and water (4.5 ml) was stirred for 2 hours at roomtemperature. After evaporation, the residue was dissolved in water (4.5ml) and the pH was adjusted to 6.5 with 6 M hydrochloride. Europium(III)chloride hexahydrate (66 mg, 0.18 mmol) in water (1.9 ml) was addedwithin 10 minutes and the pH maintained at 6.5 with solid sodiumcarbonate. After stirring the reaction for 1 hour, the pH was raised to8.5 with 1 M sodium hydroxide and the precipitate removed bycentrifugation. The filtrate was the treated with acetone, and theproduct was collected by centrifugation and washed with acetone. Theproduct was used in next step without purification. The yield was 300mg. UV(water): 339, 257 and 247 nm. ESI-TOF-MS mass for C₄₀H₃₄EuN₇O₁₀M⁻(monoisotopic): calculated 924.70, found 924.04.

Example 5

The synthesis of{2,2′,2″,2′″-{[(carboxymethyl)imino]bis(methylene)-bis{[4-[(4-isothiocyanato)phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitro)}tetrakis-(acetato)}europium(III)(5)

The compound 4 (360 mg, 0.374 mmol) in water (6.0 ml) was added slowlyto a mixture of thiophosgene (230 μl, 3.027 mmol), sodiumhydrogencarbonate (315 mg, 3,750 mmol) and chloroform (6.0 ml). Afterstirring for 1 hour, the water phase was washed twice with chloroform(2×12 ml). The pH of aqueous solution was adjusted to 7.0 with 1 Macetic acid and added acetone to water phase. The product was collectedby centrifugation and washed with acetone. The yield was 490 mg.UV(water): 333, 319 and 228 nm. ESI-TOF-MS mass for C₄₂H₃₀EuN₇O₁₀S₂M⁻(monoisotopic): calculated 1008.83, found 1007.96.

Example 6

Coupling of the chelate 5,7-dentate and 9-dentate Eu-chelates (6 and 7in Scheme 2) to protein

Labeling was performed in 10 mM borate buffer, pH 8.6–9.0 using 15- (forthe chelate 5) 50- (for the chelate 6) and 150-fold (for the chelate 7)molar excesses. Reactions were normally carried out overnight at +4° C.or at room temperature. Labeled antibodies were purified on Superfex 200HR 10/30 or Superdex 200 HiLoad 26/60 gel filtration columns (PharnaciaBiotech) using Tris-saline-azide (6.1 g/L Tris, 9.0 g/L NaCl, and 0.5g/L NaN₃), pH 7.75 as an elution buffer. The fractions containing theantibody were pooled and the europium concentrations measured against aeuropium calibrator (Innotrac Diagnostics Oy). The purified antibodyconjugate and the labeling ratio (chelates per protein) were quantifiedby calculating the protein yield or by measuring the absorbance at 280nm and subtracting the absorption caused by the added chelate.

Example 7

The Luminescence Measurements of the Chelate-labelled Antibodies

The luminescence parameters for the Eu labeled antibodies were measuredin buffer containing 5 mM Hepes, 2.1 g/L NaCl, 0.1 mM EDTA, 0.055 g/LTween20 and 1 g/L Germall II, pH 7.75. The excitation and emissionspectra, luminescence intensities and decay times were measured using aLS55 Luminescence Spectrometer (PerkinElmer Instruments), while themolar extinction coefficients (absorbance) were determined with aUV-2100 Spectrophotometer (Shimadzu). Additionally, decay times werealso measured from antibody bound to solid phase after buffer aspiratingand drying of the wells. The solid phase determinations were performedusing Cary Eclipse Fluorescence Spectrophotometer (Varian). Theluminescence measurements were standardized using 0.1 μM Eu(III) inWallac Delfia enhancement soln. (molar absortivity 37600, quantum yield70% and luminescence yield 26320). The emission intensities weremeasured using the most intense emission line at ca. 613 nm.

TABLE 1 The emission maxima (λ_(exc)), luminescence decay times (τ) andluminescence yields (ε · Φ) of Eu (III) chelates 5, 6 and 7 in proteinin Hepes buffer, pH 7.75. Chelate λ_(exc)[nm] τ[μs] ε · Φ 5 328 11208025 6 329  390 1337 7 325 1000 4787

TABLE 2 Decay times (τ) of EU (III) chelates 5, 6 and 7 coupled inprotein in various environments. Chelate τ[μs]/Hepes τ[μs]/wet surfaceτ[μs]/dry surface 5 1120 1130 1290 6  390  490 1150 7 1000 1000  990

1. A luminescent lanthanide chelate comprising a lanthanide ion and achelating ligand of formula (I)

wherein R₁ is selected from the group consisting of H, —COOH, —COO⁻,—CH₂COOH and —CH₂COO⁻; G₁ is a group consisting of one or two moieties,each moiety being selected from the group consisting of ethynediyl(—C≡C—), ethenylene (—CH═CH—), phenylene, biphenylene, naphthylene,pyridylene, pyrazinylene, pyrunidinylene, pyridazinylene, furylene,thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene,isothiazolylene, oxazolylene, isoxazolylene, furazanylene,1,2,4-triazol-3,5-ylene and oxadiazolylene; G₂ is a group capable ofbeing coupled to a biospecific binding reactant and is selected from thegroup consisting of amino, aminooxy, carboxyl, aldehyde or mercaptogroups and activated forms made of them; Z is selected from the groupconsisting of carboxyalkyl amine {—N[(CH₂)_(n)COOH] or—N[(CH₂)_(n)COO⁻]— and n=1, 2, 3,
 4. 5, or 6}, ether (—O—), thioether(—S—), carbonyl (—CO—) and unsubstituted or substituted methyl (—CR₂—)wherein group R₂ is selected from the group consisting of H, methyl,ethyl and carboxyalkyl{—CH₂)_(n)COOH or —(CH₂)_(n)COO⁻ and n=1, 2, 3, 4.5, or 6; and the lanthanide ion is a member selected from the groupconsisting of europium (III), terbium (III), dysprosium (III) andsamarium(III).
 2. The lanthanide chelate according to claim 1 wherein G₂is said activated form selected form the group consisting of isocyanato,isothiocyanato, diazonium, bromoacetamido, iodoacetamido, reactiveesters, pyridyl-2-dithio and 6-substituted4-chloro-1,3,5-triazin-2-ylamino.
 3. The lanthanide chelate according toclaim 1 wherein the chelating ligand is{2,2′,2″,2′″-{[(carboxymethyl)imino]bis(methylene)-bis{[(4-[(4-isothiocyanato)-phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}-europium(III).4. A detectable molecule which comprises a biospecific binding reactantattached to a luminescent lanthanide chelate comprising a lanthanide ionand a chelating ligand of formula (II)

wherein R₁ is selected from the group consisting of H, —COOH, —COO⁻,—CH₂COOH and —CH₂COO⁻; G₁ is a group consisting of one or two moieties,each moiety being selected from the group consisting of ethynediyl(—C≡C—), ethenylene (—CH═CH—), phenylene, biphenylene, naphthylene,pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene,thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene,isothiazolylene, oxazolylene, isoxazolylene, furazanylene,1,2,4-triazol-3,5-ylene and oxadiazolylene; G₃ is a group coupled tosaid biospecific binding reactant and is selected from the groupconsisting of thiourea (—NH—CS—NH—), aminoacetamide (—NH—CO—CH₂—NH—),amide (—NH—CO—, —CO—NH—, —NCH₃—CO— and —CO—NCH₃—), aliphatic thioether(—S—), disulfide (—S—S—) and 6-substituted-,1,3,5,-triazine-2,4-diamine;Z is selected from the group consisting of carboxyalkyl amine{—N[(CH₂)_(n)COOH]— or —N[(CH₂)_(n)COO⁻]— wherein n is 1, 2, 3, 4, 5, or6}, ether (—O—), thioether (—S—), carbonyl (—CO—) and unsubstituted orsubstituted methyl (—CR₂—) wherein group R₂ is selected from the groupconsisting of H, methyl, ethyl and carboxyalkyl [—(CH₂)_(n)COOH]— or—{(CH₂)_(n)COO⁻]— wherein n is 1, 2, 3, 4, 5, or 6]; and the lanthanideion is a member selected from the group consisting of europium (III),terbium (III), dysprosium (III) and samarium(III).
 5. The detectablemolecule according to claim 4, wherein the biospecific binding reactantis selected from the group consisting of an antibody, an antigen, areceptor ligand, a specific binding protein, a DNA probe and an RNAprobe.
 6. The detectable molecule according to claim 4, wherein thelanthanide chelate attached to a biospecific binding reactant is{2,2′,2″,2′″-{[(carboxymethyl)-imino]bis(methylene)-bis{[4-[(4-thioureylene)phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitrilo)tetrakis-(acetato)}-europium(III).7. A method of performing a biospecific binding assay, comprisinglabelling an analyte with a detectable molecule comprising a biospecificbinding reactant attached to a luminescent lanthanide chelate, therebyforming a labelled analyte; exciting said labelled analyte withradiation having an excitation wavelength, thereby forming an excitedlabelled analyte; and detecting emission radiation emitted from saidexcited labelled analyte, wherein said luminescent lanthanide chelatecomprises a lanthanide ion and a chelating ligand of formula (II)

wherein R₁ is selected from the group consisting of H, —COOH, —COO⁻,—CH₂COOH and —CH₂COO⁻; G₁ is a group consisting of one or two moieties,each moiety being selected from the group consisting of ethynediyl(—C≡C—), ethenylene (—CH═CH—), phenylene, biphenylene, naphthylene,pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene,thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene,isothiazolylene, oxazolylene, isoxazolylene, furazanylene, 1,2,4triazol-3,5-ylene and oxadiazolylene; G₃ is a group coupled to saidbiospecific binding reactant and is selected from the group consistingof thiourea (—NH—CS—NH—), aminoacetamide (—NH—CO—CH₂—NH—), amide(—NH—CO—, —CO—NH—, —NCH₃—CO— and —CO—NCH₃—), aliphatic thioether (—S—),disulfide (—S—S—) and 6-substituted-1,3,5,-triazine-2,4-diamine; Z isselected from the group consisting of carboxyalkyl amine{—N[(CH₂)_(n)COOH]— or —N[(CH₂)_(n)COO⁻] wherein n is 1, 2, 3, 4, 5, or6}, ether (—O—), thioether (—S—), carbonyl (—CO—) and unsubstituted orsubstituted methyl (—CR₂—) wherein group R₂ is selected from the groupconsisting of H, methyl, ethyl and carboxyalkyl [—(CH₂)_(n)COOH]— or—[(CH₂)_(n)COO⁻]— wherein n is 1, 2, 3, 4, 5, or 6; and the lanthanideion is a member selected from the group consisting of europium (III),terbium (III), dysprosium (III) and samarium(III).
 8. The methodaccording to claim 7, wherein the biospecific binding reactant isselected from the group consisting of an antibody, an antigen, areceptor ligand, a specific binding protein, a DNA probe and an RNAprobe.
 9. The method according to claim 7, wherein the lanthanidechelate attached to a biospecific binding reactant is{2,2′,2″,2′″-{[(carboxymethyl)imino]bis(methylene)-bis{[4-[(4-thioureylene)-phenyl]ethynyl]pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}-europium(III).